Breast cancer is a common malignancy that continues to take a devastating toll on American women. In majority of cases, breast cancer is a carcinoma arising from the transformation of mammary epithelial cells. Transformation is culmination of complex molecular genetic changes that occur in a normal cell, beginning with loss of normal growth control mechanisms. This loss of normal growth control mechanisms usually results in bypass of cellular senescence, and immortalization. The molecular genetic changes that result in senescence bypass and immortalization in human mammary epithelial cells (HMECs) are largely unknown. This proposal focuses on a candidate oncogene, Bmi-1 that has been shown to modulate senescence mechanisms in fibroblasts and cooperate with c-Myc in tumorigenesis. Recently, we observed that Bmi-1 can induce telomerase, cause significant replicative life-span extension and induce immortalization of HMECs. We also found that Bmi-1 is overexpressed in several breast cancer cell lines. Although, Bmi-1 is a known oncogene, its role in telomerase induction and breast cancer is not known. Our purpose to identify the molecular mechanisms of telomerase induction and immortalization by Bmi-1, and study its role in breast cancer. Using HMECs and wild type or mutant Bmi-1, first we will define the role of RING finger domain of Bmi-1 in immortalization and telomerase induction. Next, we will perform structural and functional analysis of telomerase promoter to identify the sequences required for telomerase induction in Bmi-1 overexpressing cells. Our third aim is to study the role of Bmi-1 interacting proteins, clone and identify novel Bmi-1 interacting proteins that may be involved in telomerase regulation. Next, we will study the role of Bmi-1 in ubiquitination and proteolysis of important cell cycle regulatory proteins and tumor suppressors. Finally, we will examine the oncogenic potential of Bmi-1 in-vitro using cell culture studies, and in-vivo using breast tumors at different stages to determine if Bmi-1 is overexpressed at a particular stage of breast cancer. These studies will define the role of Bmi-1 in breast cancer and determining the diagnostic and prognostic value of Bmi-1 expression as an early marker of breast cancer. These studies will also help us in understanding the mechanism of telomerase regulation and may yield new targets for telomerase inhibition.